Photodynamic therapy (PDT) is a fast developing modality for the diagnosis and treatment of both oncological and nononeological diseases and it involves the use of photochemical reactions mediated though the interaction of photosensitizing agents, light, and oxygen for the treatment of malignant or benign diseases. PDT is a 2-step procedure. In the first step, the photosensitizer is administered to the patient by one of several routes (eg. topical, oral, intravenous), and it is allowed to be taken up by the target cells. The second step involves the activation of the photosensitizer in the presence of oxygen with a specific wavelength of light directed toward the target tissue. Because the photosensitizer is preferentially absorbed by hyperproliferative tissue and the light source is directly targeted on the lesional tissue, PDT achieves both selectivity, minimizing damage to adjacent healthy structures. References may be made to Lane, N. Scientific American 2003, 38, 45; Bonnett, R. Chem, Soc. Rev. 1995, 24, 19; Dougherty, T. J. Photochem. Photobiol. 1987, 45, 879; Kessel, D. Dougherty, T. J. Phorphyrin Photosensitization; Plenum Publishing Corp. New York, 1983; Bissonette, R.; Bergeron, A.; Liu, Y. d. J Drugs. Dermatol. 2004, 3, 26-31; Jeffes, E. W.; McCullough, J. L.; Weinstein, G. D.; Fergin, P. E.; Nelson, J. S.; Shull, T. F. Arch. Dermatol, 1997, 133, 727-732. The process requires the presence of a photosensitizing agent, which is capable of being taken up by target tissues and which, on irradiation by light of a particular wavelength, generates highly reactive species which are toxic to those tissues. Photodynamic therapy has advantages over many other conventional therapies due to the selectivity of the photodynamic process. There is more sensitizer in the tumor tissues than in the normal tissues; this reduces the potential for destruction of normal tissues. In addition the ability to direct light specifically onto the target cells and tissues by the use of fiber-optic technology further increased the selectivity of this process. Furthermore, the use of photosensitizing agents, which produce no response until irradiated with light, significantly reduces the potential for side effects. References may be made to Jeffes, E. W.; McCullough, J. L. Weinstein, G. D.; Kaplan, R.; Glazer, S. D.; Taylor, J. R. J. Am. Acad. Dermatol, 2001, 45, 96-104; Kurwa, H. A.; Yong-Gee, S. A.; Seed, P. T.; Markey, A. C.; Barlow, R. J. J. Am. Acad. Dermatol, 1999, 41, 414-8; Pariser, D. M.; Lowe, N. J.; Stewart, D. M,; Jarratt, M. T.; Lucky, A. W.; Pariser, R. J. J. Am. Acad. Dermatol, 2003, 48, 227-32.
In PDT, the detection of tumor tissue (diagnosis) is equally important when compared to the destruction via either apoptosis or necrosis of tumor cells (treatment). Near-infrared (NIR) dyes are presently attracting considerable interest as fluorescence probes for the detection of cancer. References may be made to Lin, Y.; Weissleder, R.; and Tung, C. H. Bioconjugate Chem, 2002 13, 605-610; Achilefu, S.; Jimenez, H. N.; Dorshow, R. B.; Bugaj, J. E.; Webb, E. G.; Wilhelm, R. R.; Rajagopalan, R.; Johler, J.; Erion, J. L. J. Med. Chem. 2002 45, 2003-2015; Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A. S. Bioconjugate Chem. 1996, 7, 356-362. Since tissue is relatively transparent to NIR light, near infrared fluorescence imaging (NIRF) and PDT are capable of detecting and treating, respectively, even subsurface tumors. In this context the present invention aims at the development of efficient NIR absorbing fluorescent probes based on porphyrins for biological applications. We have synthesized porphyrin based molecules which exhibit absorption and emission in the NIR region and have substituents like hydroxyl and glycolic groups, which would render them amphiphilicity thereby increasing their solubility, fluorescence intensity and accelerating their cellular uptake.
In a diagnostic technique, a dye is administered and allowed to distribute in the body as in the case of the treatment technique. However, in addition to the tumor selectivity, the sensitizer in the diagnostic technique should exhibit significant fluorescence yields under physiological conditions. Hence the development of photosensitizers, which have strong absorption in the long wavelength region, non-toxic to normal tissues, soluble in buffer at physiological pH, and exhibit higher therapeutic efficacy are still desired. Also the design of functional molecules that can target specific cancer cells are extremely important because of the biochemical and biomedical applications.
Porphyrin molecules are one of the photosensitizers currently being investigated. Porphyrins are macrocyclic molecular compounds with a ring-shaped tetrapyrrolic core. References may be made to Mahler, H. R.; Cordes, E. H. Biological Chemistry, 2d ed. 1966, 418; Joni, G.; Reddi, E. Int J Biochem, 1993, 25, 1369-75. Wiehe, A.; Shaker, Y. M.; Brandt, J. C.; Mebs, S.; Senge, M. O. Tetrahedron, 2005, 61, 5535-5564; and Pushpan, S. K.; Venkatraman, S.; Anand, V. G.; Sankar, J.; Parameswaran, D.; Ganesan, S.; Chandrashekar, T. K. Curr. Med. Chem.—Anticancer Agents, 2002, 2, 187-207. As such, porphyrins are commonly found in their dianionic form coordinated to a metal ion. The unique properties of the tetrapyrrolic core have made porphyrin central in many biological systems that play a vital role in many life processes. Several compounds which are critically important for essential biological processes, such as chlorophyll and heme, are derived from the coordination of a metal ion with a porphyrin nucleus. Porphyrins are able to form metal chelates with a large variety of metal ions, including: cobalt, copper, iron, magnesium, nickel, silver, and zinc. Heme is an iron chelate of a porphyrin, while chlorophyll and bacteriochlorophyll are magnesium chelates. Porphyrin such as these is generally synthesized from the precursors glycine and suceinyl CoA. References may be made to L. Stryer, Biochemistry, 2nd ed. 504-507 (1981). It has further been well established that the hydro- or Lipo-philicity of a photosensitizes strongly affects the binding of the photosensitizer to a target cell, and as a consequence, its cytotoxic activity. References may be made to Merehat et al., J. Photochem. Photobiol. B: Biol., 35:149-157 (1996).
Currently known porphyrin based photosensitizers includes hematoporphyrin derivative (HpD) and photofrin called the first generation photosensitizers. HpD is facing the major drawbacks includes (a) it is a mixture of at least nine components, (b) preparation is highly sensitive to reaction conditions and (c) causes cutaneous photosensitivity. Another example of porphyrin based photosensitizer is 5, 10, 15, 20-tetrakis (meta-hydroxyphenyl)-chlorin which is commercially known as foscan. The current methods of their synthesis, and known techniques for their use are inadequate for many intended applications. References may be made to Konan, Y. N.; Cerny, R.; Favet, J.; Berton, M.; Gurny, R.; A lleman, E. Eur. J. Pharm. Biopharm. 2003, 55, 115-124; and Nawalany, K.; Rusin, A.; Kepczynki, M.; Mikhailov, A.; Kramer-Marek, G.; Snietura, M.; Poltowicz, J.; Krawcyzk, Z.; Nowakowska, M. J. Photochem. Photobiol. B: Biology, 2009, 97, 8-17. This is true in part due to the need for high concentrations of reagent and the requirement of extended irradiation periods. These factors render the methods burdensome and inconvenient. In addition, such conditions are not suitable for many medical and/or industrial applications. It is thus seen that there is a need for novel photosensitizing agents for medical or other applications. It would be an improvement in the art to provide an agent that utilizes a pathway for inactivating or killing an to organism which is non-mutagenic. Finally, it would be an additional improvement in the art to provide such a photosensitizer that is capable of functioning effectively at lower concentrations and over shorter periods than those currently known and taught in the art.
Our interest in this area originated from the idea of utilizing the derivatives of currently existing porphyrin derivatives for photodynamic applications. In recent years a great variety of non-porphyrinic sensitizers are being developed for use in PDT. Methylene blue, a red-light absorbing phenothiaxinium dye, has previously been used extensively as a biological assay stain and can be used in the clinical diagnosis of a variety of diseases and as a tumor marker in surgery. However, its use as an in vivo photosensitizer is limited by its reduction by ubiquitous cellular enzymes to the colorless form, which is photodynamically inactive. Rhodamine is an important laser dye and is being used in red light emitting laser dyes for a long time now. Because of their specific uptake by mitochondria and their known use as a biochemical fluorescent probe, rhodamine classes of molecules are being used as sensitizers in the treatment of malignant tumors. But on the other hand the readily available commercial dye, rhodamine 123 is a poor phototoxin because of its high fluorescence quantum yield, which leads to a low triplet quantum yield. References may be made to Yamamoto, H.; Okunaka, T.; Furukawa, K.; Hiyoshi, T,; Konaka, C.; Kato, H. Curr. Sci, 1999, 77, 894; Sharman, W. M.; Allen, C. M.; van Lier, J. E.; Drug Discovery Today, 1999, 4, 507; Milgrom, L.; MacRobert, S; Chem. Britain, 1998, 45; Bonnett, R. Chem. Soc. Rev, 1995, 24, 19. Dolphin, D. Can. J. Chem, 1994, 72, 1005.
Another class of molecules developed from our group for use in PDT is squaraines. Squaraines are a class of dyes possessing sharp and intense absorption in the near infrared region and exhibit significant triplet quantum yields. Among the various squaraine dyes developed, bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine is found to be a potential photosensitizer certainly possesses clinical applications though in vitro and in-vivo experiments. The dye is found to be a promising compound in PDT as an effective NM photosensitizer. References may be made to U.S. Pat. No. 6,770,787 B2; Ramaiah, D.; Joy, A.; Chandrasekar, N.; Eldho, N. V.; Das, S.; George, M. V; Photochem. Photobio, 1997, 65, 783-790; Ramaiah, D.; Eckert, 1.; Arun, K. T.; Weidenfeller, L.; Epe, B. Photochem. Photobiol, 2002, 76, 672-677.
Although there are several non-porphyrinic photosensitizers are available, the fact that a naturally occurring dye such as porphyrin is the drug of choice in PDT has prompted the search for better photosensitizers based on porphyrin macrocycle. These tetrapyrrole rings form a class of dyes possessing sharp and intense absorption bands in the visible to near infra red region. The photophysical and photochemical properties of these have been studied extensively, because their absorption and photochemical characteristics make them highly suitable for a number of biological and industrial applications. References may be made to U.S. Pat. No. 4,649,151; R. Bonnet, R. D. White, U. J. Winfield, M. C, Berenbaum. Biochem. J., 1989, 261, 277-280; D. Kessel. Photochem. Photobiol., 1984, 39, 851-859.
The novel porphyrin derivatives of the general formula 1 claimed in the current patent application are derivatives of tetraphenyl porphyrin. Preliminary investigations by us indicated that substitution of more number of hydroxy groups to the porphyrin meso-phenyl ring results in their increased solubility in the aqueous medium and enhanced intersystem crossing efficiency, when compared to the currently existing one. When compared to other porphyrins, which are not water soluble, the derivatives claimed in the present invention exhibit high water solubility.
More over, insertion of heavy metals like Zinc onto the porphyrin macrocycle also caused increased intersystem crossing efficiency and hence high singlet oxygen generation efficiency. These dyes exhibited absorption in the range from 400-700 nm and fluorescence emission in the range 600-800 nm. These dyes are having almost good fluorescence quantum yields in the range 0.15-0.23. Also the quantum yields of triplet excited states (□T) of these porphyrin derivatives are found to be in the range 0.60-0.75 and the quantum yields of singlet oxygen generation (□(1O2)) in the range 0.40-0.75, depending on the nature of the substituent present on the mesa phenyl ring and the inserted metal. The cytotoxicity, mutagenicity, kinetics of uptake and release and the cellular localization studies of these porphyrin derivatives using mammalian cell lines and bacterial strains indicated that these dyes exhibit significant cytotoxicity upon excitation with visible light and the mechanism of their biological activity could be attributed to the in vitro generation of singlet oxygen.
Cellular localization studies of the synthesized porphyrin derivatives showed that they are preferentially accumulating on the nucleus with red fluorescence during visible light excitations. Hence these derivatives can be used as NIR fluorescent probes for nuclear staining. Most of the reported porphyrins such as chlorin e6 and to hematoporphyrin derivatives localize mainly at the plasma membrane, whereas, our photosensitizers that localize at the nucleus would be much more effective in producing photodynamic damage.
Cellular uptake studies on the porphyrin derivatives of the present invention showed that they are far more efficient. The novel porphyrin derivatives of the present invention show maximum cellular uptake within 10 min, which has been evidenced through the fluorescence microscopic images given as FIG. 10 while the cellular uptake of PhtofrinR is maximum only within 4 h.
Also the present investigation showed that the porphyrin derivatives have binding affinity towards proteins such as serum albumin and hence can be used as NIR fluorescent probes for protein labeling.
In the present invention we have synthesized novel porphyrin derivatives and demonstrated their, potential as NIR PDT agents and fluorescent probes for biological and biochemical applications.